Association of Ovar‐DRB1 alleles with innate immune responses in sheep

Abstract Background Major histocompatibility complex (MHC) is the best characterised genetic region associated with adaptive immune responses, including humoral and cell‐mediated immunities. Objectives In this study, the association of MHC class II alleles with inflammatory cytokines and acute‐phase proteins was evaluated in sheep population. Methods Allelic diversity of second exon of ovine DRB1 locus (Ovar‐DRB1.2) was determined in 100 indigenous Iranian Lori‐Bakhtiari fat‐tailed sheep using restriction fragment length polymorphism and direct sequencing methods. The association of DRB1.2 alleles with inflammatory cytokines (interleukin‐1β, IL‐1β; IL‐6 and tumour necrosis factor‐α) and acute‐phase proteins (serum amyloid A, alpha‐1‐acid glycoprotein and haptoglobin) was examined using generalised linear model and multivariate regression analysis. Results Seven distinct RsaI restriction patterns and fourteen alleles were identified in this population. Allele DRB1*2101 showed a negative influence on the IL‐6 response and was associated with lower serum level of IL‐6. DRB1.2 heterozygous individuals also showed higher haptoglobin concentration than homozygotes. Conclusions These results provide additional support for the association between Ovar‐DRB1 alleles and regulation of immune responses in sheep population. Description of MHC polymorphism and its role in the controlling of immune responses will increase our understanding of host–pathogen interactions, and ultimately facilitate the selection of disease‐resistant flocks in genetic breeding programs.

bind and present extracellular pathogens to the circulating helper T lymphocytes and initiate humoral immunity (Abbas et al., 2010). Class II loci have been found to be highly polymorphic and considered as a principal target in genotyping studies aimed to evaluate the association of MHC with immune responses and phenotypic traits (Ali et al., 2019;Ashrafi et al., 2014;Cinar et al., 2016;Larruskain et al., 2010;Shen et al., 2014).
MHC in domestic sheep (Ovis aries) is known as "Ovar" and physically located on chromosome 20 between bands q15-q23 (Mahdy et al., 1989). The structure of MHC in sheep is similar to the other mammalian species, including three main classes (I, II and III), each having different functional roles (Amills et al., 1998). Among the Ovar class II genes, DRB1 is highly polymorphic and more than 100 alleles have been identified in the second exon of this locus (Ovar-DRB1.2) encodes the antigen-binding cleft of MHC molecules (Ballingall et al., 2008;Ballingall & Tassi, 2010;Gruszczyñska et al., 2005;. Polymorphism in this exon has offered possibilities for effective immune responses against variety of pathogens Larruskain et al., 2010Larruskain et al., , 2012Nagaoka et al., 1999). Research on the MHC as a candidate marker for disease resistance has become a major focus in breeding strategies during the recent years. Numerous investigations have indicated the MHC polymorphism and its involvement in genetic resistance to diseases in different sheep populations (Ali et al., 2019;Herrmann-Hoesing et al., 2008;Larruskain et al., 2012;Li et al., 2010;Stear et al., 2006). Correlation of MHC with some production and reproduction features, such as growth, body weight and fertility has also been documented in sheep (Ashrafi et al., 2014;Cinar et al., 2016;Gruszczyńska et al., 2000).
Determining the role of MHC in controlling the immune responses will provide great information for selection of disease-resistant population in genetic breeding programs. However, effects of phenotypic traits including immune responses are not consistent across the breeds, and selection based on a specific marker should be applied with great caution. In order to determine the variation in disease resistance or susceptibility among the populations, it is important to characterise the MHC polymorphism and define its relationship to immune responsiveness in each breed. In this study, Ovar-DRB1.2 genetic diversity was evaluated in an Iranian indigenous sheep breed and the association of DRB1 alleles with innate immune responses, including inflammatory cytokines and acute-phase proteins, was also investigated in this population. Although correlation of Ovar-DRB1 alleles with adaptive immune responses against infectious diseases has been demonstrated in sheep population, no evidence of MHC relation to innate immunity has been reported in sheep.

Sampling and DNA extraction
One hundred blood samples were collected from six-month-old indigenous Iranian fat-tailed ewes, belonged to the Lori-Bakhtiari breed, with normal delivery and similar grazing history. Lori-Bakhtiari breed is originated from Chaharmahal and Bakhtiari province in the west of Iran and kept mainly for meat production.

Assessment of innate immune responses
Innate immune responses were assessed by measuring the quantitative level of inflammatory cytokines and acute-phase proteins in the serum samples. Serum levels of interleukin-1β (IL-1β), IL-6, tumour necrosis factor alpha (TNF-α), serum amyloid A (SAA), alpha-1-acid glycoprotein (AGP) and haptoglobin (Hp) were measured by quantitative sandwich enzyme immunoassay method using commercial sheep-specific kits (Shanghai Crystal Day Biotech, Shanghai, China).

Data analysis
Population genetic analysis including the number of alleles, allele and genotype frequencies, observed and expected homozygosity and heterozygosity for Ovar-DRB1.2 locus were estimated using Popgene software (Yeh et al., 1997). Deviation of population from Hardy-Weinberg equilibrium (HWE) was also assessed using likelihood ratio test. Unbiased expected heterozygosity and the number of alleles were applied to evaluate the amount of gene diversity in this population (Nei, 1973).
In order to evaluate the possible association and interaction between Ovar-DRB1.2 alleles and inflammatory factors, generalised linear model (GLM) and multivariate regression analysis were used.
Serum level of inflammatory cytokines and acute-phase proteins were considered as response variables that were specific for each animal.
Presence or absence of DRB1.2 alleles in each individual was fitted as a fixed factor and alleles with frequency less than 5% were not included in the model. As immune responses are probably influenced by age and type of birth, these parameters were also fitted as covariates in all of the

Ovar-DRB1.2 genotyping
PCR-RFLP analysis identified 7 distinct RsaI restriction patterns (a, b, c, d, f, g and h) and 22 genotypes at DRB1.2 locus in Lori-Bakhtiari population. Pattern g had the highest (20.5%) and pattern h the lowest (1.0 %) frequency. Genotype ga was the most (12%) and dd and ca the least (1%) frequent genotypes in this population. Ovar-DRB1.2 sequencing data also revealed 14 alleles in this locus (Table 1). A high level of heterozygosity (82%) and good genotype frequency fit to the HWE was observed in Lori-Bakhtiari population (p = 0.26) ( Table 2).

Association of Ovar-DRB1.2 alleles with innate immune responses
Serum level of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and acute-phase proteins (SAA, AGP and Hb) were measured by quantitative ELISA and presented in Table 3

DISCUSSION
Having an effective natural defence system is essential for controlling the potential pathogens and ensuring the animal health (Linde et al., 2008). Exposure to pathogens activates the signalling pathways in innate immune cells and results in the production and secretion of three major inflammatory cytokines, including IL-1, IL-6 and TNF-α. Under the influence of inflammatory cytokines, especially IL-6, hepatocytes increase the production of acute-phase proteins such as C-reactive protein, serum amyloid A, alpha-1-acid glycoprotein, haptoglobin, sialic acid and ceruloplasmin. These inflammatory mediators trigger the innate immune responses by recruiting sentinel cells including macrophages and neutrophils to the sites of inflammation, promoting phagocytosis, removal of dead and damaged cells and activating the complement system (Tizard, 2013). Abbreviations: AGP, alpha-1-acid glycoprotein (g/l); Hb, haptoglobin (g/l); IL-1β, interleukin-1β (pg/ml); IL-6, interleukin-6 (pg/ml); SAA, serum amyloid A (μg/ml); TNF-α, tumour necrosis factor-alpha (pg/ml).
Immune system mechanisms, including innate and adaptive immune responses, are controlled by the influence of multiple genes along with the additive effect of environmental factors. Because of the low heritability and difficulties associated with a reliable evaluation of these traits in animal populations, breeding strategies based on the direct selection of improved immune responses are practically impossible (Boonyanuwat et al., 2006;Notter, 1999). Compared to the traditional selective breeding methods, marker-assisted selection program attempts to combine the information of genetic markers and quantitative trait loci with the phenotypic data, in order to improve the selection responses. In this program, a trait of interest would be selected based on a genetic marker that is linked to the genes controlling this trait. In other words, genetic markers are used to indicate the presence of a specific phenotypic trait (Ribaut & Ragot, 2007;Wakchaure et al., 2015). Considering the strong correlation between MHC haplotypes and resistance or susceptibility to a wide range of diseases in different animal species, MHC can be considered as a candidate genetic marker for immune responsiveness in breeding strategies.
In this study, the Ovar-DRB1. showed a significant correlation with serum total protein, total globulins, α2-globulin, β-globulin and γ-globulin. The findings of this study indicated that the mutation in DRB1.2 region had a considerable impact on immunity, and to a less extent, on production traits (Zamani et al., 2016). Most relevant studies on Ovar polymorphism and disease resistance and/or susceptibility have mainly focussed on DRB1 locus and significant associations have been reported, the majority being concerned with gastrointestinal nematodes and cestodes (Ali et al., 2019;Shen et al., 2014;Valilou et al., 2015). Allele DRB1*1101 has been associated with drastic reduction in faecal egg counts following infection with Teladorsagia circumcincta (Ali et al., 2019). A strong correlation between DRB1 alleles and hydatidosis (cystic echinococcosis) resistance and susceptibility has also been reported in different sheep populations (Li et al., 2010;Shen et al., 2014). Likewise, there are a few reports on the involvement of Ovar-DRB1 alleles in resistance to bacterial and viral diseases including footrot (Escayg et al., 1997;Valilou et al., 2016), Johne's disease (Reddacliff et al., 2005), ovine progressive pneumonia (Herrmann-Hoesing et al., 2008), bovine leukaemia , Maedi-Visna (Larruskain et al., 2010) and pulmonary adenocarcinoma (Larruskain et al., 2012). However, this study is the first research attempted to link the Ovar-DRB1 alleles to the innate immune responses in sheep.

CONCLUSION
Use of genetic markers such as MHC for selecting improved immune responses has shown the promising results in the breeding programs.
In the present study, moderate genetic diversity and high level of heterozygosity were observed at the DRB1 locus in indigenous Lori-Bakhtiari population. Allele DRB1*2101 was negatively associated with the IL-6 response in the studied population. However, further investigations are necessary to determine whether this allele contains the causative mutations or is only a marker in linkage disequilibrium with a causative variant. Understanding the indigenous breeds' genetic patterns of MHC and their associations with immune responses seems to be worthwhile with respect to the conservation and genetic improvement of sheep populations.

ACKNOWLEDGEMENTS
This work was funded by School of Veterinary Medicine, Shiraz University, grant no. 96GRD1M271548. We also express our appreciation to Prof. Gholamreza Nikbakht Brujeni for helping with statistical analysis of the data. visualisation.

ETHICAL APPROVAL
The Animal Care and Use Committee of the Shiraz University approved the experimental procedures which are in compliance with the regulations for protection of animal research.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.